1. Field of the Invention
The present invention relates to a method of producing large-scale quantities of biologics. In particular, the present invention relates to a scaled-up process of manufacturing a primary cell derived biologic.
2. Description of Related Art
There are various methods in the art used to produce biologics from cells which generally involve the steps of stimulating cells through incubation and washing cells to obtain the desired product.
For example, U.S. Pat. No. 4,390,623 to Fabricius discloses a serum-free and mitogen-free T-cell growth factor (interleukin-2) preparation prepared from human, bovine, or porcine peripheral mononuclear blood cells which are washed several times with a liquid tissue culture medium and then stimulated in tissue culture medium supplemented with serum and mitogen. The separated stimulated cells are again washed with fresh tissue culture medium to remove substantially all of the serum and mitogen. The washed cells are suspended in fresh tissue culture medium and conditioned under incubation conditions to transfer the growth factor into the liquid. The tissue culture medium separated from the stimulated cells can be recycled to stimulate additional cells. The supernatant can be concentrated from 50 to 100-fold on an ultrafilter.
U.S. Pat. No. 4,406,830 to Fabricius discloses a process for producing serum-free, mitogen-free Interleukin-1 (II-1) (also known as lymphocyte activating factor LAF) and serum-free, mitogen-free II-2 by incubating peripheral mononuclear blood (PBL) cells in a serum-free liquid tissue culture medium to remove residual serum proteins on the surfaces of the PBL cells, activating the incubated cells with a mitogen, washing the activated cells with a sterile liquid to remove the mitogen from the cells and conditioning the serum-free mitogen-free activated cells in a liquid tissue culture medium to produce a serum-free, mitogen-free Interleukin-1 (IL-1), contacting the IL-1 containing liquid tissue culture medium with novel blood serum glycoprotein, and incubating the cells in the presence of IL-1 and the novel blood serum glycoprotein to induce synthesis of IL-2 and to transfer the IL-2 (T-cell growth factor) from the cells to the liquid phase of the tissue culture medium to thereby produce a serum-free, mitogen-free IL-2.
U.S. Pat. No. 5,503,828 to Testa discloses a method of large-scale production of alpha interferon through induction and purification. A mixture of alpha interferon subtypes produced from peripheral blood leukocytes is produced by (a) preparing human peripheral blood leukocytes by collecting buffy coats and lysing red blood cells with ammonium chloride; (b) suspending leukocytes at a cell density of 1−10×106 cells/ml in an induction medium, comprising Eagle's MEM containing Earle's Salts, L-glutamine, non-essential amino acids, 4.46 mg/ml Tricine, pH 7.4, 24 μg/ml neomycin sulfate, vitamins B3 and/or C, sodium bicarbonate, and between 0.1 to 1.5 mg/ml human agamma serum; (c) adding crude or purified alpha interferon as a primer to the leukocytes suspended in the induction medium; (d) incubating the suspension for a sufficient time at about 36 degrees C. while stirring at 100-300 rpm; (e) adding between 50-500 hemagglutinin units per ml of Sendai virus to the suspension; (f) incubating for a sufficient time at about 36 degrees C. while stirring at 100-300 rpm; (h) centrifuging at about 2,500 rpm to remove cells and debris; and (i) collecting crude alpha interferon as product, without ever separating one alpha interferon subtype from the other subtypes present in the alpha mixture.
U.S. Pat. No. 6,350,589 to Morris discloses a method of producing multisubtype Type 1 interferons. The method includes the steps of (a) culturing leukocytes; (b) stimulating the leukocytes to produce a crude interferon; (c) concentrating the crude interferon to remove low-molecular weight contaminants; (d) liquid volume to produce a concentrated crude interferon; (e) removing a substantial amount of serum albumin and other contaminants from the concentrated crude interferon to produce a partially purified interferon mixture containing a plurality of subtypes; (f) removing substantially all remaining serum albumin and other contaminants from the partially purified interferon mixture to generate an interferon mixture having a purity of between about 50% and about 80%; and (g) purifying the about 50% to about 80% interferon mixture to produce a highly purified mixture of Type I interferon having a purity of at least about 95% and containing no more than about 35% by weight IFN.alpha.-2 and IFN .alpha.-8 subtypes.
U.S. Pat. No. 6,896,879 to Talor discloses a method of producing a cytokine mixture that is serum-free, mitogen-free, and antibiotic-free. In the manufacturing process, mononuclear cells are separated from human donor “buffy coats” by step-gradient centrifugation and cultured with phytohemagglutinin (PHA) to enhance production and secretion of IL-2 and other cytokines from the donor white blood cells in culture. Subsequently, the culture supernatant is aseptically harvested, clarified and subjected to a commercial virus exclusion process. The supernatant is then further concentrated approximately 10 fold by ultrafiltration and microfiltration. At this point, Human Serum Albumin, Inj. USP is added and the concentrate is then buffered to a physiological pH and brought to a target IL-2 concentration per the label claim (example 400 IU/mL). The concentrate is then subjected to a second micro-filtration (0.22 micron-rated filter) and aseptically dispensed into sterile serum-type vials and labeled by its IL-2 content. Product potency is measured by the incorporation of radio-labeled thymidine by a cytotoxic T-lymphoid line (CTLL-2). The final injectable agent is further tested by ELISA for the presence of five marker cytokines: IL-2, IL-1β, GM-CSF, IFN-γ, and TNF-α.
U.S. Pat. Nos. 5,632,983; 5,698,194; 6,977,072; 7,153,499; 7,182,942 to Hadden disclose a method of producing a natural cytokine mixture (NCM) that is a unique cytokine mixture of IL-1β, IL-2, IL-6, IL-8, INF-γ, and TNF-α. Buffy coat white cells of human blood from multiple HIV-negative hepatitis virus-negative donors are collected. The cells from the donors are pooled and layered on ficoll hypaque gradients (Pharmacia) to yield lymphocytes free of neutrophils and erythrocytes. In a preferred embodiment for the production of NCM lymphocytes are washed and distributed in X vivo-10 media (Whittaker Bioproducts) to flasks (MicroCELLector™ T-25 Cell Culture Flasks) in which are immobilized stimulants, i.e. mitogens. The immobilization process for the stimulants is as described by the manufacturer for immobilizing various substances for panning procedures, i.e. separating cells, in the flasks. The cells are incubated for 24-48 hours in X vivo-10 media with 80 μg/ml ciprofloxacin (Miles Lab) at 37 degrees C. in a CO2/air incubator. Following incubation the supernatants are poured off and collected. Human serum albumin (HSA) can be added to stabilize the interleukins. Generally the HSA is used at 0.1 to 0.5% (weight by volume). The supernatants are stored at 4 degrees C. to −70 degrees C. The pooled supernatants are characterized by measuring the cytokine content by bioassay for IL-2 and ELISAs for one or more of the interleukins IL-1-IL-15, CSFs, TNFs, and IFNs. Sterility is tested by culture in thioglycolate broth and endotoxin measured by limulus lysate assay as is known in the art. Each supernatant is standardized either by concentration or amount administered so that comparisons can be made. In particular the IL-2 equivalence for each supernatant is utilized. DNA and virus exclusion, if used, employs such techniques as ultrafiltration, ethanol fractionation, polyethylene glycol/bentonite precipitation, and/or solvent/detergent treatment as has been used for intravenous gamma globulin (IGIV News Update brochure). Photochemical inactivation, aluminum phthalocyanine, or gamma irradiation can be used. This process is further discussed in the present invention below.
There are several limitations of manual processes used for producing biologics such as operator sensitivity, potential for contamination in an open system, inconsistent ratios and total protein levels in the final product, all of which make the product unsuitable for pharmaceutical grade production. To deal with these problems in the past, cumbersome procedures were performed such as filters, starch, manual centrifugations, and washes. Previous processes were bench top procedures that produced inconsistent batches and small-scale quantities of product.
Another step in biologics processing that must be considered is the removal of viruses. Patient safety is paramount, and in biotechnology processes there is a risk of adventitious viruses contaminating the incoming cells. Accordingly, inactivation and removal steps are sought to remove viruses that may or may not be present. Several logs of clearance/inactivation are required, per FDA and ICH guidances. Regulatory agencies suggest testing of the unprocessed bulk for potential viruses as well including in the process methods which provide a minimum of 4 log10 of virus inactivation/removal to be considered significant. It is suggested the methods include two (or more) orthogonal steps preferably with one targeting non-enveloped viruses. The regulatory guidance suggests that validation studies should be conducted to characterize the ability of production methods to remove/inactivate adventitious viruses exhibiting a range of biochemical and biophysical properties to characterize the robustness of the process.
For primary cell derived biologic production donor leukocytes, source cells for cytokine production, are screened by the blood centers for presence of viral nucleic acid by PCR (NHCV and NHIV) and traditional viral antigens (human immunodeficiency virus (HIV), hepatitis C(HCV), hepatitis B (HBV) and human T-lymphotropic virus (HTLV)). However other viruses, Epstein Barr (EBV), Cytomegalovirus (CMV) and Human Parvovirus B-19 (B-19) may still be present in qualified donors and used for production. Detectable levels of EBV could be present in up to 100% of healthy donors (Walling et. al., 2003). B-19 levels in asymptomatic individuals have been reported to be greater than 1012 per mL (Doyle and Corcoran, 2006) and infection results in a brief period of viraemia with titers as high as 1014 per mL (Anderson 1985). Due to the extensive cell washing used in the primary cell derived biologic process, most plasma associated viruses are essentially removed from the donor leukocytes, and any virus detected virus in the primary cell derived biologic bulk, prior to downstream removal/inactivation steps, would only be those released from infected cells.
Nevertheless, robust inactivation/removal processes are required to assure product safety.
The manual production process for the primary cell derived biologic is labor intensive and not readily amenable to scale-up and is limited to volumes of sterile fluid which could be handled by a manual process. Therefore, process development is sought to reduce manual manipulations, and achieve practicality for commercialization. Furthermore, a virus inactivation method is desired.